Abstract

Early and sensitive identification of stress, infectious disease and/or tissue trauma facilitates timely initiation of prophylactic measures and/or treatment whether they be elimination of the insult or supportive therapy. A variety of reagents and techniques were developed to assess the immunologic health of cetacean species, with preliminary application being primarily directed at killer whales. This immunologic evaluation included immunophenotyping peripheral blood leukocytes, identification of alterations in leukocyte adhesion proteins, assessment of lymphocyte function and identification of blood-borne mediators of inflammation. Immunologic evaluation of leukocyte phenotype and adhesion proteins required generation of species-specific monoclonal antibodies that could recognize cetacean leukocyte differentiation antigens. Antibodies were developed that could distinguish B lymphocytes (human homologues of CD19 and CD21), T lymphocytes (human homologue of CD2) and naive versus memory T lymphocytes (CD2 vs human homologue of CD45R). Antibodies specific for adhesion proteins included the human homologue of CD11/18 and two antibodies that recognize adhesion proteins (referred to as D and F6B) for which we have not yet identified a human or murine counterpart. The classic lymphocyte blastogenesis assay was modified to permit identification of minor perturbations in lymphocyte function by utilizing both optimal and sub-optimal concentrations of T and B lymphocyte mitogens (Concanavalin A, Con-A; Pokeweed mitogen, PWM; Phytohemagglutinin, PHA). Techniques were developed for cryopreservation of mononuclear peripheral blood leukocytes that could be stored in liquid nitrogen for future functional analysis. This approach permitted greater standardization of the assay as all bloods were stored at about 10°C for 24 hr prior to cryopreservation, minimizing variation due to differences in shipment time of blood from the site of collection to the laboratory. Identification of inflammation was based upon the detection of interleukin 6 (IL-6) in plasma or serum; IL-6 is the primary mediator of the acute phase response. This latter measurement of inflammation is the subject of a companion abstract by C. Funke, et al.

Our current database demonstrates that both stress and infection can be detected, but not necessarily differentiated, utilizing this combination of immunologic tools. However, identification of either condition facilitates early treatment whether it be removal or reduction of the stressor and/or initiation of supportive therapy. While data is still preliminary, statistical analysis would suggest that combining such an immunologic analysis with classical CBC and clinical chemistry data would substantially widen our window into the health monitoring of cetacean species. Furthermore, the current approach of generating baseline data on individual animals creates an individual fingerprint that permits identification of subtle perturbations that would otherwise go undetected if we were to rely on average values obtained for the species as a whole. Similar approaches to monitoring the immunologic health of valuable animals on a routine basis would be easily justified.